A molecular complex of Cav1.2/CaMKK2/CaMK1a in caveolae is responsible for vascular remodeling via excitation–transcription coupling

Significance Excitation–transcription (E-T) coupling can initiate and modulate essential physiological or pathological responses in cells, such as neurons and cardiac myocytes. Although vascular myocytes also exhibit E-T coupling in response to membrane depolarization, the underlying molecular mechanisms are unknown. Our study reveals that E-T coupling in vascular myocytes converts intracellular Ca2+ signals into selective gene transcription related to chemotaxis, leukocyte adhesion, and inflammation. Our discovery identifies a mechanism for vascular remodeling as an adaptation to increased circumferential stretch.

nylon suture (Natsume Seisakusho) (5). This creates high pressure in the middle vessel. Control vessels were second-order mesenteric arteries obtained along the intestine, remote from the ligated vessels. The mesenteric artery was isolated 2d, 7d or 14 d after surgery. To evaluate the effects of STO609 in vivo, mice were treated with STO609 (1 mg/kg) intraperitoneally on the day of surgery (at least 2 hours before surgery) and then three time per week. STO609 was dissolved in DMSO at 10 mg/mL and stored at -20°C until usage. This stock solution was diluted to 0.2 mg/mL with sterile PBS. A PBS solution containing 2% DMSO was used as a vehicle control.

ELISA
The P-CREB levels in arteries were quantified using ELISA. Two types of ELISA detecting (i) phosphorylated CREB at S133 (KHO0241, Thermo Fisher Scientific) and (ii) total CREB (KHO0231, Thermo Fisher Scientific) were performed using arterial lysates according to protocols provided by the company. Second and third-order mesenteric arteries were flash-frozen in liquid N2. The frozen samples were pulverized and proteins were extracted using a cell lysis buffer (100 µL) containing 10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate (Thermo Fisher Scientific). Protease inhibitor cocktail (MilliporeSigma) and 1 mM PMSF (TCI) were added just prior to experiments. The lysates were sonicated briefly and centrifuged to remove debris. The supernatant was diluted to 1:20 and subjected to ELISA. Both phosphorylated and total CREB amounts of each sample were calculated from standard curves, and the relative P-CREB level to total CREB of each sample was obtained.

Histological analysis
Isolated arteries were fixed in 4% PFA, embedded in paraffin, and cut into 5-μm thick sections for routine hematoxylin-eosin (HE) staining (Biopathology Institute). Medial thickness was measured at 4 different points and averaged per sample. Image analysis was performed using NIS Elements (Nikon).

Transfection using virus vectors
Cav1 (NM_001753) and CaMKK2 (XM_006530233) was labeled with mCherry or GGECO1.1 (Addgene plasmids #32445) at the N termini. pCMV-PV-NLS-GFP was obtained from Addgene (#17300). These constructs were cloned into pENTR1A and obtained pAd/CMV/V5-DEST or BacMam pCMV-DEST vectors using Gateway system (Thermo Fisher Scientific). Adenovirus and baculovirus vector were harvested from 293A and Sf9 cells after introducing linearized pAd/CMV/V5-DEST and bacmid, respectively. Adenovirus was used for knock-in or overexpression experiments (Fig. 2C, 4D and 5J-L). On the other hand, baculovirus was utilized for TIRF imaging (Fig. 3) because of relatively low expression efficiency compared with adenovirus and high signal to noise ratio. Primary vascular myocytes at semiconfluent condition were infected for 12 hours and then washed. Experiments were performed 48-72 hours after infection.

Real-time qPCR.
Total RNA extraction, reverse transcription and real-time quantitative PCR were carried out as previously reported (6). The sequence of primers was listed in Table S2. For normalization of gene expression among each samples, Ppia and Hprt were utilized as endogenous housekeeping genes because these expression was similar between 5 mM and 60 mM K + conditions. Averaged Cq value of these genes was used for calculation.

RNA sequence analysis
RNA was extracted from samples using TRIzol and PureLink RNA spin columns (Thermo Fisher Scientific). Genomic DNA was digested using TURBO™ DNase (Thermo Fisher Scientific). Conventional RNA-Seq was performed by Novogene. The RNA-seq reads were aligned with the mouse reference genome mm9 using TopHat2 software. Prior to differential gene expression analysis, the read counts were adjusted for each sequenced library by edgeR program package through one scaling normalized factor. Differential expression analysis of two conditions was performed using the DEGSeq R package. The P values were adjusted using the Benjamini & Hochberg methods. Corrected qvalue of 0.005 and |log2 (Fold Change)| of 1 were set as the threshold for significantly differential expression.

TIRF imaging
Two-dimensional single-molecular imaging were done using a TIRF imaging system (Nikon), which consists of a fluorescent microscope (Ti2; Nikon), objective lens (CFI Plan Apo TIRF 60× or 100×; Nikon), EM-CCD camera (C9100-12; Hamamatsu Photonics), and NIS Elements software (Nikon) (1, 6). For co-localization analysis, two molecules were labeled with green or orange indicators, and corresponding fluorescence puncta having higher intensity than background levels were converted to binary images. Then, co-localized puncta were extracted by image arithmetic operations using the NIS Elements software. The ratio of the number of co-localized puncta to the total number of puncta was calculated. For GGECO imaging, fluorescence signals from GGECO1.1 are described as F/F0, where F is the sum fluorescence intensity within the regions of interest (ROI, 2 µm square) in the TIRF area during measurements, and F0 is the baseline F value obtained as the average intensity of the ROI for 5 sec before stimulation. GGECO1.1 puncta whose maximal increase in fluorescence intensity due to depolarization stimulus was higher than 5×SD of baseline values were accepted as positive GGECO1.1 puncta. Images were collected at an interval of 800 msec for two color imaging, i.e. GG-CaMKK2 and mCh-cav1 or 100 msec for single color imaging, i.e. GG-CaMKK2 only. Immunolabeling images were collected by exposure for 100 msec. The resolution of images was 270 or 160 nm per pixel (x − y) and less than 200 nm (z). All experiments were performed at room temperature.

Confocal imaging
Confocal images were obtained using a laser scanning confocal fluorescent microscope (A1R, Nikon) equipped with a fluorescent unit (ECLIPSE Ti), objective lens (Plan Apo 60× or 20×), and NIS Elements software (Nikon). For P-CREB analysis, fluorescence signals from secondary antibodies binding to P-CREB primary antibodies and nuclear indicators (Hoechst or TO-PRO3) were converted into binary images. Next, fluorescence signals of nuclei co-localized with P-CREB signals were extracted by image arithmetic operations using the NIS Elements software. The ratio of the number of nuclei co-localized with P-CREB signals to the total number of nuclei was calculated. For each artery, three image sections (215 µm×215 µm) were acquired and the calculated ratio values were averaged.

Ca 2+ imaging
Intracellular Ca 2+ imaging was performed using a confocal microscope. For monitoring [Ca 2+ ]i, mesenteric artery myocytes were incubated with 10 μM Fluo-4/AM for 20 min at room temperature. The excitation and emission wavelength were 488 nm and 500-530 nm, respectively. When the amplitude of [Ca 2+ ]i elevation in primary vascular myocytes of WT and cav1-KO cells were recorded, cells were loaded with 10 μM Fluo-4/AM and 10 μM Fura Red/AM for 30 min to perform ratio metric measurement. These indicators were excited at 488 nm and emission was collected at 500-530 nm (Fluo-4) and 662-737 nm (Fura-Red). Fratio was calculated by dividing fluorescence intensity of Fluo-4 by that of Fura-Red. When Ca 2+ responses of myocytes transfected with PV-NLS-GFP were recorded, cells were loaded with 6.7 µM CaSiR/AM (Goryo Chemical) and Hoechst. These indicators were excited at 640 and 405 nm and emission was collected at 662-737 nm (CaSiR) and 425-475 nm (Hoechst). ROIs corresponding to whole cell regions and nuclei were created according to the fluorescence of CaSiR and Hoechst, respectively. The sum of CaSiR intensity and area corresponding to cytosol were calculated by subtracting these values of nuclei from those of whole cell regions.

In situ PLA
To clarify whether cav1/Cav1.2/CaMKK2/CaMK1a co-localizes within 40 nm in arterial myocytes, a PLA was performed using a PLA kit (Duolink, MilliporeSigma) (6). Myocytes were fixed with 4% paraformaldehyde in PBS and treated with 0.2% Triton X-100. Cells were then labeled with primary antibodies mentioned above at 4°C for 12 h. After washing repeatedly, cells were incubated in a humidified chamber at 37°C for 1 h with secondary antimouse PLUS and anti-rabbit MINUS PLA probes and then washed in Duolink Wash Buffer A. The preparations were incubated in ligase solution at 37°C for 30 min in a humidifier chamber and then washed repeatedly in Wash Buffer A. Samples were incubated in Amplification Polymerase solution at 37°C for 100 min in a humidifier chamber and then washed repeatedly in Duolink Wash Buffer B. Fluorescence images were observed using a confocal imaging system. When two PLAs probes were within 40 nm, positive signals (green puncta) were generated. The excitation of fluorescent puncta was illuminated at 488 nm. Negative control experiments were performed using myocytes treated with either of antibodies. PLA puncta were extracted by binary image processing and the relative area of these puncta to the whole cell area was calculated.

Data notation and statistical analysis
Pooled data are shown as the mean ± SEM. The significance of differences between two groups was evaluated using the two-tailed t test after the application of the F test. Data from more than two groups were compared using one-way or two-way analysis of variance (ANOVA) followed by Tukey or Dunnett tests. In all cases, p values <0.05 were considered to be significant. All data were obtained from at least three independent experiments.

Data Availability.
The datasets generated in this study are available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE183588 Supplementary Figures   Fig. S1. Expression profiles of CaMK family genes in mesenteric artery and primary vascular myocytes. (A and B) Splice variants of CaMK2γ (Camk2g, A) and CaMK2δ (Camk2d, B) in primary vascular myocytes, mesenteric artery (MA), and brain were identified using reverse-transcription PCR analysis. Negative control data was obtained by adding water instead of cDNA (non-template control, NTC). In both Panel (A) and (B), predicted band size for each splice variant is indicated within the box below. Representative photos from three independent experiments are shown.

Fig. S2. Effects of CaMK inhibitors on 60 mM K + superfusate-induced [Ca 2+ ]i elevation by Ca 2+ influx through voltage-dependent Ca 2+ channels in mesenteric artery myocytes. (A)
Traces of fluorescence signals from Fluo-4/AM in mesenteric artery myocytes. Myocytes were stimulated by 60 mM K + solution twice, and drugs were applied at the second 60 mM K + stimulation. (B) Relative Fluo-4 intensity normalized to untreated control (the first 60 mM K + stimulation). Data were obtained from 19-43 cells from three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey test (**p<0.01).   PLA was performed to seek evidence for direct coupling of cav3 with Cav1.2 and CaMKK2 in primary myocytes. WT cells labeled with only the cav3 antibody were used for a negative control. Each data set was obtained from 9-18 cells. Statistical analysis was performed using one-way ANOVA followed by Tukey test (**p<0.01 vs. WT cav3). PLA was performed to seek evidence for direct coupling of CaMK1a with cav1, CaMKK2 and Cav1.2 in mesenteric artery myocytes from WT mice. To obtain negative control data, myocytes were treated only with the anti-CaMK1a antibody and then subjected to PLA. Each data set was obtained from 14-19 cells. Statistical analysis was performed using one-way ANOVA followed by Dunnett test (*p<0.05 vs. CaMK1a).

Fig. S7. Endothelium-denuded mesenteric arteries can exhibit depolarization-induced gene transcription very similar to the pattern obtained in intact arteries.
Endothelium of mesenteric artery beds were removed by perfusing 0.2% sodium deoxycholate (SD) dissolved in ice cold PBS for 120 sec. (A) Confocal images of endothelial layers were obtained from control (treated with PBS) and SD groups (9 arteries from 3 mice per group). Vessels were stained with Hoechst (left). The density of endothelial cells (ECs) is compared (right). Note that the number of ECs was reduced, and ECs exhibited morphologic abnormality i.e. round, not elliptic, shape (indicated by arrowheads), and these ECs often aggregated in the SD group. For each artery, three image sections (215 µm×215 µm) were acquired and the calculated EC density values were averaged. (B) The gene expression of an endothelium marker, Von Willebrand Factor (VWF) was compared by qPCR. Data were obtained from 5 mice per group. (C) Acetylcholine (ACh)-induced vasodilation was recorded in before (Control) and after SD treatment. Vasocontraction was induced by 30 mM K + Hanks solution and then arterial beds were dilated by 1 µM ACh. ACh-induced vasodilation (%) was estimated from ACh-induced reversal of KCl-mediated vasocontraction. Data were obtained from 5 mice. (D) Gene transcription after depolarizing stimulus for 1 hour was confirmed by qPCR. Data were obtained 5 mice per group. Statistical analysis was performed using two-tailed t test (**p<0.01).

Fig. S8. Sustained increase in transmural pressure in mesenteric artery causes CREB phosphorylation in vascular myocytes by activating the Cav1.2/CaMKK2/CaMK1a axis.
The possibility that increased perfusion pressure can cause CREB phosphorylation was addressed using ex vivo model. (A) Mesenteric artery preparations were perfused with 5 mM K + solution at 0, 40 and 80 mmHg for 30 min, and then fixed and stained with P-CREB antibody. The effect of the L-type Ca 2+ channel blocker nicardipine (Nic) on 80 mmHg-induced CREB phosphorylation also examined. The P-CREB positive myocyte ratio in vivo was estimated using acute fixation by perfusing ice cold 4% PFA from heart under deep anesthesia. Data were obtained from 9-16 arteries. (B) The effect of CaMK inhibitors KN93 and STO609 on 80 mmHg-induced CREB phosphorylation was examined. Data were obtained from 16-20 arteries. (C) High pressure (80 mmHg) was applied to mesenteric artery of WT and cav1-KO mice. Mesenteric artery treated with methyl-β-cyclodextrin (MβCD) was also utilized. Data were obtained from 15 arteries per group. Statistical analysis was performed using one-way (A) or two-way ANOVA (B and C) followed by Tukey test (**p<0.01).

Fig. S9. Changes in cross-section area (CSA) of mesenteric arteries preloaded with high pressure. (A)
Changes in CSA of second-order mesenteric arteries from WT (left) and cav1-KO (right) mice 2, 7 and 14 days after ligation. Control vessels were second-order mesenteric arteries located along the intestine, remote from the ligated vessels. Data were collected from 4-7 arteries. (B) Changes in CSA of second-order mesenteric arteries from mice treated with DMSO (left) or STO609 (right) 2, 7 and 14 days after ligation was plotted. Data were collected from 5-6 arteries. Statistical analysis was performed using two-way ANOVA followed by Tukey test (*p<0.05, **p<0.01; #p<0.05, ##p<0.01 vs. HP group at the same day).